Abstract Neural circuit function requires synaptic inhibition mediated by ?-aminobutyric acid (GABA). Glutamine is the major metabolic precursor for neuronal GABA synthesis and is supplied to neurons via the activity of the astrocyte-specific enzyme glutamine synthetase (GS). Inhibition or brain-specific genetic ablation of GS leads to impaired GABAergic inhibition, intractable epilepsy, and death. Consistent with this, deficits in GS expression are found in the brains of patients and animal models of epilepsy. Deficits in GS levels have also been reported in the brains of patients with Alzheimer's disease and schizophrenia. Despite the critical role GS plays in neuronal physiology there have been no systematic studies to evaluate how GS activity is regulated in the mammalian brain to meet the demand of neurons for glutamine. GS structure is highly conserved through evolution, but all mammalian isoforms contain unique amino acids that are potential substrates for phosphorylation which are adjacent to, or within the catalytic site of this enzyme. These residues include threonine residue 301 (T301) and serine residue 343 (S343), which are consensus sites for a phosphorylation by a variety of classical 2nd messenger dependent protein kinases including cAMP-dependent (PKA) kinase. However, it remains to be determined if GS is a phospho-protein in the brain and if this covalent modification impacts on enzyme activity and/or stability. To address this issue, we will use stable isotope labeling of amino acids in culture (SILAC) coupled with liquid chromatography-tandem mass spectrometry (LC- MS/MS) to 1) identify sites of phosphorylation, and 2) quantify their stoichiometry of phosphorylation under basal conditions and after the activation of PKA in situ, in vitro and in vivo. To complement our LC-MS/MS studies we will use recently developed phospho-specific antibodies to further analyze GS phosphorylation. In parallel, we will assess the effects phosphorylation have on GS activity and stability. Finally, we will determine if seizure activity induced by chemico-convulsants modifies GS phosphorylation and activity in the mouse brain. Preliminary studies have allowed us to formulate a central hypothesis that will be tested in this proposal; GS activity is inhibited by PKA-dependent phosphorylation of T301/S343, and enhanced phosphorylation of these residues contributes to the deficits in GS activity seen during epilepsy. Our proposal will center on the following specific aims: Aim 1. To test the hypothesis that PKA mediated phosphorylation of GS leads to decreased enzyme activity. Aim 2. To test the hypothesis that ?phospho-dependent? inactivation of GS is enhanced by seizure like activity. Collectively these studies will provide the first evidence of the mechanism by which astrocytes regulate GS activity. Therefore, they may lead to the development of novel therapeutic strategies to increase GS activity to alleviate the burdens of epilepsy and neurodegenerative disorders.